Method for preparing peptide fragments, kit for preparing peptide fragments to be used therein, and analysis method

ABSTRACT

A method of proteolyzing a protein, including immobilizing a protein in at least one pore of a porous body, and contacting the protein immobilized in the pore and a protease immobilized on a solid surface such that the protease selectively accesses a site of the protein and proteolyzes the protein at the site.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of International ApplicationNo. PCT/JP2013/074292, filed Sep. 9, 2013. The entire contents of thisapplication are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Intention

The present invention relates to a method for preparing peptidefragments by site-selectively proteolyzing a protein, such as anantibody, using a protease, and a kit for preparing peptide fragments tobe used therein. Further, the present invention relates to a method foranalyzing peptide fragments, prepared by the method, by massspectrometry or the like to detect or quantitate a protein.

Discussion of the Background

A method has been developed in which a protein to be analyzed issubjected to site-selective proteolysis to reduce the number (of types)of peptide fragments in a sample to improve the accuracy of analysis andsimplify the process of analysis. For example, WO 2008/079914 proposes amethod in which an antibody is subjected to pepsin digestion to producean F(ab′)₂ fragment, and then the F(ab′)₂ fragment is further digestedwith a protease such as trypsin to produce peptide fragments containingthe complementarity-determining region (CDR) of the antibody, and thepeptide fragments containing CDR are detected and quantitated by massspectrometry. JP 2011-130749 A reports that combined use of pepsin orpapain and a specific ion improves the efficiency of proteolysis withsuch a protease.

Fei Xu et. al., Anal. Chem., 2010, 82, 10045-10051 report an example inwhich the efficiency of trypsin digestion of albumin is increased byallowing an albumin solution to pass through a nylon porous membranehaving trypsin immobilized in pores thereof. Qianhao Min et. al., Chem.Commun., 2010, 46(33), 6144-6146 report that a protein having a smallmolecular weight can be selectively subjected to trypsin digestion byusing mesoporous silica having trypsin immobilized in pores thereof.

Hereinafter, the structure of an antibody will be described. Allantibodies have two heavy chains (H chains) and two light chains (Lchains). One light chain and one heavy chain are linked through adisulfide (S—S) bond to form a heterodimer, and the two heterodimers arefurther linked through two disulfide bonds to form a “Y”-shapedheterotetramer (see FIG. 2). An antibody has one Fc (Fragment,crystallizable) domain comprising heavy chains and two Fab (Fragment,antigen binding) domains comprising a heavy chain and a light chain, andthe Fc domain and the Fab domains are linked through a hinge region.

The Fc domain of an antibody mainly has the function of initiating areaction after the antibody binds to an antigen (effector function), andmost of antibodies derived from the same species have a common aminoacid sequence in the Fc domain. On the other hand, the end (on theN-terminal side) of the Fab domain has the function of binding to anantigen. The N-terminal part of the Fab domain diversely changes in itsamino acid sequence so as to be able to bind to various antigens. Thisregion is called variable region (V region), and the variable region ofthe light chain and the variable region of the heavy chain are called VLregion and VH region, respectively. The Fab and Fc domains other thanthe V region are called constant region (C region) that varies little inamino acid sequence. The constant region of the light chain is called CLregion, and the constant region of the heavy chain is called CH region.The CH region is further divided into three regions, CH1 region, CH2region, and CH3 region. The Fab domain of the heavy chain comprises theVH region and the CH1 region, and the Fc domain of the heavy chaincomprises CH2 and CH3. The hinge region is located between CH1 and CH2.

The specificity (i.e., specific bindability to an antigen) of anantibody is determined by the combination of amino acid sequences of theV region. The light chain and the heavy chain each have threecomplementarity-determining regions (CDRs) in the V region of the Fabdomain. CDR is also called hypervariable region, and varies in aminoacid sequence depending on the type of antibody. There are 3 CDRs oneach of the heavy and light chains of an antibody (6 types of CDRs intotal), which creates diversity that allows the antibody to bind tovarious antigens. In other words, CDRs are regions characterizing anantibody, and therefore an antibody can be identified by identifying theamino acid sequences of CDRs thereof.

As described above, the Fab domains and Fc domain of an antibody arelinked through a hinge region. Papain which is a kind of proteaseproteolyzes the hinge region, and therefore two Fab domains and one Fcdomain are produced by papain digestion of an antibody. Further, pepsinwhich is a kind of protease proteolyzes one of the two disulfide bonds,i.e., the Fc domain-side (C-terminal side) disulfide bond of the hingeregion, and therefore an F(ab′)₂ domain having two Fab domains linkedtogether and many Fc domain fragments are produced by pepsin digestion.

SUMMARY OF THE INVENTION

According to one aspect of the present invention, a method ofproteolyzing a protein includes immobilizing a protein in at least onepore of a porous body, and contacting the protein immobilized in thepore and a protease immobilized on a solid surface such that theprotease selectively accesses a site of the protein and proteolyzes theprotein at the site.

According to another aspect of the present invention, a kit forproteolyzing a protein includes a porous body having pores in which aprotein is to be immobilized, and particles having an average particlediameter larger than an average pore diameter of the pores, wherein theparticles have a surface on which a protease has been immobilized or tobe immobilized.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 is a conceptual diagram for illustrating the principle ofsite-selective proteolysis according to an aspect of the presentinvention.

FIG. 2 is a schematic diagram for illustrating the structure of anantibody.

FIG. 3 is a schematic diagram of one embodiment of a kit for preparingpeptide fragments.

FIG. 4 shows electrophoretic patterns obtained in an experiment forexamining the quantitative ratio of a protease.

FIG. 5 shows mass spectra (MALDI-TOFMS) obtained in the experiment forexamining the quantitative ratio of a protease.

FIG. 6 shows mass spectra (MALDI-TOFMS) obtained in an experiment forexamining proteolysis time.

FIG. 7 shows the result of database analysis based on the result of massspectrometry of tryptic fragments of trastuzumab.

FIG. 8 shows a mass spectrum (MALDI-TOFMS) of tryptic fragments oftrastuzumab. The fragments shown in FIG. 8 include FTISADTSK (residues68-76 of SEQ ID NO: 1), IYPTNGYTR (residues 51-59 of SEQ ID NO: 1),DTYIHWVR (residues 31-38 of SEQ ID NO: 1), WGGDGFYAMDYWGQGTLVTVSSASTK(residues 99-124 of SEQ ID NO: 1), LLIYSASFLYSGVPSR (residues 46-61 ofSEQ ID NO: 2), and ASQDVNTAVAWYQQKPGK (residues 25-42 of SEQ ID NO: 2).

FIG. 9 shows chromatograms of LC-MS analysis of tryptic fragments oftrastuzumab. The fragments shown in FIG. 9 include YADSVK (residues60-65 of SEQ ID NO: 1), FTISADTSK (residues 68-76 of SEQ ID NO: 1),IYPTNGYTR (residues 51-59 of SEQ ID NO: 1), GLEWVAR (residues 44-50 ofSEQ ID NO: 1), DTYIHWVR (residues 31-38 of SEQ ID NO: 1), andASQDVNTAVAWYQQKPGK (residues 25-42 of SEQ ID NO: 2).

FIG. 10 shows mass spectra (LS-MS) of tryptic fragments of trastuzumab.The fragments shown in FIG. 10 include YADSVK (residues 60-65 of SEQ IDNO: 1), FTISADTSK (residues 68-76 of SEQ ID NO: 1), IYPTNGYTR (residues51-59 of SEQ ID NO: 1), GLEWVAR (residues 44-50 of SEQ ID NO: 1),DTYIHWVR (residues 31-38 of SEQ ID NO: 1), and ASQDVNTAVAWYQQKPGK(residues 25-42 of SEQ ID NO: 2).

FIGS. 11A and 11B show the amino acid sequences of heavy and lightchains of trastuzumab, respectively, wherein peptide fragmentsidentified by mass spectrometry in this experiment are underlined. FIG.11A shows SEQ ID NO: 1, and FIG. 11B shows SEQ ID NO: 2.

FIG. 12 shows electrophoretic patterns obtained in an experiment forstudying mixed protease proteolysis.

FIG. 13 shows mass spectra (MALDI-TOFMS) obtained in the experiment forstudying mixed protease proteolysis.

DESCRIPTION OF THE EMBODIMENTS

The embodiments will now be described with reference to the accompanyingdrawings, wherein like reference numerals designate corresponding oridentical elements throughout the various drawings.

In a method for preparing peptide fragments according to an aspect ofthe present invention, a substrate protein to be proteolyzed isimmobilized in pores of a porous body, and the porous body having thesubstrate protein immobilized thereon is brought, in a liquid, intocontact with fine particles having a protease immobilized on a surfacethereof. FIG. 1 is a conceptual diagram for illustrating the principleof protease proteolysis according to an aspect of the present invention.

On the surface of fine particles 10 (average particle diameter D₁), aprotease 15 is immobilized. A porous body 20 has a plurality of pores 29(average pore diameter D₂), and a substrate protein 25 is immobilized inthe pores. In the method according to an aspect of the presentinvention, as described above, both the protease 15 and the substrateprotein 25 are immobilized on solid phases in a small region, andprotease proteolysis is performed by contact between the solid phases.

The average particle diameter D₁ of the fine particles 10 is larger thanthe average pore diameter D₂ of the porous body 20. Therefore, the fineparticles 10 can access the shallow parts (opening portions) of thepores 29 and their vicinity, but cannot access the deep parts of thepores 29. As a result, the protease 15 immobilized on the surface of thefine particles 10 cannot access the deep parts of the pores 29. In FIG.1, a dotted line near each of the pores 29 indicates the limit of theregion accessible to protease 15.

In this way, the accessibility of the protease 15 to the substrateprotein 25 in the pores 29 is site-selectively limited so that therelative probability of accessibility of the protease 15 to the liquidphase side (“Y”-shaped site in FIG. 1) of the substrate proteinincreases. This makes it possible to subject the substrate protein 25 tosite-selective protease proteolysis to obtain peptide fragments.

Substrate Protein

The substrate protein 25 is a protein to be analyzed. The type of thesubstrate protein is not particularly limited. However, from theviewpoint of performing site-selective proteolysis, the substrateprotein preferably has a molecular diameter larger than that of theprotease 15. The substrate protein may be a protein complex. As themolecular diameter, a value determined based on structural analysis byX-ray or NMR is available from various documents or databases. Forexample, the molecular diameter of an antibody is about 15 nm.Alternatively, the molecular diameter may be determined by, for example,X-ray small angle scattering or may be roughly estimated from amolecular weight. As reference examples, Table 1 shows molecular weightsand molecular diameters of proteins used as marker molecules todetermine the separation properties of an ultrafiltration membrane.

TABLE 1 molecular molecular protein weight [Da] diameter [nm] sucrose340 1.1 raffinose 590 1.3 vitamin B12 1,360 1.7 bacitracin 1,410 1.7insulin 5,700 2.7 cytochrome C 13,400 3.8 myoglobin 17,000 4.0α-chymotrysinogene 25,000 4.6 pepsin 35,000 5.0 ovalbumin 43,000 5.6Bovine albumin 67,000 6.4 aldolase 142,000 8.2 γ-globulin 150,000 8.4

The substrate protein 25 is preferably one that can site-specificallybind into the pores 29 of the porous body 20. Site-specific binding ofthe substrate protein 25 allows a site other than the binding site to besubjected to selective protease proteolysis. For example, a proteinbearing, at its C- or N-terminus, a tag sequence such as a His tag (tagpeptide containing about 6 continuous histidine residues) or abiotinylated peptide, an enzyme that specifically binds with a specificsubstrate, or the like may also be used as the protein that cansite-specifically bind.

In an aspect of the present invention, an antibody is particularlypreferably used as the substrate protein that can site-specifically bindinto the pores of the porous body. Immobilization of the Fc domain ofthe antibody on the porous body 20 allows the Fab domain of the antibodyto be subjected to selective protease proteolysis. Although the type ofthe antibody is not particularly limited, a monoclonal antibody ispreferred. Examples of the monoclonal antibody include: human antibodiessuch as panitumumab (Vectibix), ofatumumab (Arzerra), golimumab(Simponi), and ipilimumab (Yervoy); humanized antibodies such astocilizumab (Actemra), trastuzumab (Herceptin), bevacizumab (Avastin),omalizumab (Xolair), mepolizumab (Bosatria), gemtuzumab ozogamicin(Mylotarg), palivizumab (Synagis), ranibizumab (Lucentis), certolizumab(Cimzia), ocrelizumab, mogamulizumab (Poteligeo), and eculizumab(Soliris); and chimeric antibodies such as rituximab (Rituxan),cetuximab (Erbitux), infliximab (Remicade), and basiliximab (Simulect).These antibodies are used as antibody drugs (molecularly-targeteddrugs), and the concentrations of the antibodies in blood need to bequantitated in clinical trials or the like.

In an aspect of the present invention, the Fab domain of the monoclonalantibody can be subjected to site-selective protease proteolysis toobtain peptide fragments, and the antibody can be identified andquantitated by mass spectrometry of the obtained peptide fragments. Theanalysis method is a method in which peptide fragments derived from thevariable region of the antibody are detected to identify (detect) orquantitate the antibody, that is, a method in which peptide fragmentsderived from the antibody are directly measured. Therefore, in thisaspect, the analysis method requires no specific binding substance suchas an antigen, and therefore can be applied irrespective of the type ofantibody. Therefore, the method can be applied not only to theabove-mentioned antibodies but also to newly-developed monoclonalantibodies.

Porous Body

The material of the porous body 20 is not particularly limited as longas the material has pores 29. Although the pores shown in FIG. 1 have asemi-spherical shape, the shape of the pores is not particularlylimited. A porous body having through-holes, such as a porous membrane,may also be used.

For the porous body 20, activated carbon, a porous membrane, porousresin beads, metal particles, or the like may be used. Among them, onethat can specifically bind with the substrate protein is preferred, andone that can site-specifically bind with the substrate protein isparticularly preferred. For example, affinity column packing beads usedto purify a specific protein or the like can be satisfactory.

The porous body 20 preferably used in an aspect of the present inventionis one in which a linker molecule 21 that can site-specifically interactwith the substrate protein 25 is immobilized in the pores 29 thereof.Examples of the interaction between the substrate protein and the linkermolecule include chemical binding, hydrogen binding, ion binding,complex formation, hydrophobic interaction, van der Waals interaction,electrostatic interaction, and stereoselective interaction.

The optimum linker molecule can be appropriately selected, depending onthe type or binding site of the substrate protein, from functionalgroups such as an amino group, a carboxyl group, and an epoxy group;labeling compounds such as biotin and digoxygenin; proteins such asavidin, streptoavidin, Protein A, Protein G, and immunoglobulin; variousligands; substrate compounds for enzymes; silica; and metal chelates.

Protein G, Protein A, or the like is preferably used as the linkermolecule 21, when the substrate protein 25 is an antibody. Protein A orProtein G site-specifically binds with the Ig loop in Fc domain of theantibody. The use of the porous body 20 having the linker molecule 21,such as Protein A or Protein G, immobilized in the pores 29 allows theFc domain of the antibody (substrate protein 25) to be site-specificallyimmobilized in the pores so that the Fab domain of the antibody islocated on the liquid-phase side (near the opening portions of thepores). Such immobilization of the antibody in the pores in a givendirection controls the orientation of the antibody in the pores, andtherefore the Fab domain can be site-selectively proteolyzed with theprotease.

Further, when the substrate protein is immobilized in the pores so as tobe present in a small environment at the interface between the solidphase and the liquid phase, the substrate protein is likely to bedenatured and molecular fluctuations are disturbed so that theprobability of being attacked by the protease increases. Further, in anaspect of the present invention, the protease is immobilized on theparticles, and therefore an environment is created in which the proteaseis sterically stable and autolysis is less likely to occur. This isconsidered to increase the stability of the protease. Therefore, in themethod according to an aspect of the present invention, site-selectiveprotease proteolysis can be performed, and in addition, high activity ofthe protease can be maintained.

The size of the pores 29 of the porous body 20 is not particularlylimited. The size of the pores is preferably determined in considerationof the molecular diameter of the substrate protein etc. so that the tipof the substrate protein, i.e., the site to be selectively proteolyzed,is located near the opening portions of the pores 29 when the substrateprotein 25 is immobilized. The average pore diameter D₂ of the porousbody 20 is appropriately set to fall in the range of, for example, about10 nm to 500 nm and to be smaller than the average particle diameter D₁of the fine particles 10. The average pore diameter D₂ of the porousbody 20 is, for example, preferably about 20 nm to 200 nm, morepreferably about 30 nm to 150 nm. Particularly, when the substrateprotein 25 is an antibody, in order to immobilize the Fc domain of theantibody in the pores to subject the Fab domain of the antibody tosite-selective proteolysis, the pore diameter of the porous body ispreferably 30 nm to 150 nm, more preferably 40 nm to 120 nm, furtherpreferably 50 nm to 100 nm.

The size of the linker molecule is selected in consideration of the sizeof the pores or size of the substrate protein so that the selectiveproteolysis site of the substrate protein is located near the openingportions of the pores. The size of a molecule in which the linkermolecule binds with the substrate protein is preferably about 0.5 to 1.5times, more preferably about 0.6 to 1.2 times, further preferably about0.7 to 1.1 times, particularly preferably about 0.8 to 1 times the porediameter of the porous body. When the linker molecule is not immobilizedon the porous body 20 and the substrate protein directly binds into thepores of the porous body, the molecular diameter of the substrateprotein and the pore diameter of the porous body preferably satisfy theabove relationship.

Immobilization of Substrate Protein

A method for immobilizing the substrate protein 25 in the pores 29 ofthe porous body 20 is not particularly limited, and an appropriatemethod can be adopted depending on the properties of the substrateprotein and the porous body (or the linker molecule immobilized on theporous body) etc. For example, when the porous body has Protein A orProtein G immobilized in the pores thereof, an antibody can be easilyimmobilized in the pores by mixing a suspension of the porous body and asolution containing the antibody.

The quantitative ratio between the porous body and the substrate proteincan be appropriately set depending on the purpose. For example, in thecase of quantitative analysis of the substrate protein, it is desiredthat almost entire amount of the substrate protein in a sample should beimmobilized on the porous body. Therefore, the quantitative ratio ispreferably set so that the amount of the porous body becomes higher thanthe estimated amount of the substrate protein contained in the sample.

Protease

The protease 15 recognizes the amino acid sequence of the substrateprotein and selectively proteolyzes a specific bond in a specificsequence. In an aspect of the present invention, the substrate protein25 is immobilized in the pores 29 of the porous body 20, and theprotease 15 proteolyzes the substrate protein 25 at a specific aminoacid sequence site, so that peptide fragments are obtained.

Examples of the protease include trypsin (which proteolyzes a peptide atthe C-terminal side of basic amino acid residues (Arg and Lys)), lysylendopeptidase (which proteolyzes a peptide at the C-terminal side of aLys residue), arginine endopeptidase (which proteolyzes a peptide at theC-terminal side of an Arg residue), chymotrypsin (which proteolyzes apeptide at the C-terminal side of aromatic amino acid residues (Phe,Tyr, and Trp)), V8 protease (which proteolyzes a peptide at theC-terminal side of a Glu residue), pepsin, and papain. Two or more ofthese proteases may be used in combination.

When peptide fragments of the substrate protein after proteaseproteolysis are subjected to mass spectrometry as a measurement sample,the protease to be used is preferably one with low autolysis and highselectivity for a sequence to be proteolyzed. When acommercially-available protease is used, a mass spectrometry-gradeprotease or a sequencing-grade protease is preferably used. For example,native trypsin derived from a living body has low specificity for aproteolysis site because pseudo trypsin that exhibits chymotrypsin-likeactivity is generated due to autolysis. Therefore, massspectrometry-grade trypsin is commercially available which achieves highresistance to autolysis due to reductive methylation of lysine residuesof trypsin.

In order to improve the site-selectivity of protease proteolysis of thesubstrate protease, it is important to limit the region where theprotease can access the substrate protein. Therefore, the moleculardiameter of the protease is preferably smaller than that of thesubstrate protein. More specifically, the molecular diameter of theprotease is preferably 10 nm or less, more preferably 8 nm or less,further preferably 6 nm or less, particularly preferably 5 nm or less. Aprotein having a molecular weight of about 30 kDa, such as trypsin orlysyl endopeptidase, has a molecular diameter of about 4 nm (see Table 1shown above).

Among the above-mentioned proteases, trypsin is particularly preferablyused in an aspect of the present invention. As described above, trypsinhas a small molecular diameter and its active site is present inside itsmolecule. This limits the region where the active site can access thesubstrate protein, which makes it possible to improve thesite-selectivity of protease proteolysis. Particularly, when thesubstrate protein is an antibody, the protease to be used is preferablytrypsin.

In proteome analysis study, digestion with a combination of trypsin andlysyl endopeptidase has attracted attention in recent years as atechnique for improving the recovery rate of peptide fragments (J.Proteome Res., 2012, 11(11), 5145-5156). The reason for this isconsidered to be that trypsin has the property of allowing adecomposition reaction to proceed in stages from the outside of a stericstructure, and lysyl endopeptidase first proteolyzes mainly the hingeregion of an antibody. In an aspect of the present invention, on theother hand, it is preferred that trypsin is used alone or that even whenlysyl endopeptidase or the like is used in combination with trypsin, theamount of trypsin is preferably 90% or higher of the total amount ofproteases used, in order to suppress the proteolysis of the hinge regionof an antibody and to selectively proteolyze the Fab domain (morepreferably, the V region of the Fab domain) of the antibody.

Fine Particles

The fine particles 10 are used for the purpose of immobilizing theprotease 15 on the surface thereof to control the accessibility of theprotease to the substrate protein 25 immobilized in the pores 29 of theporous body 20. Therefore, the average particle diameter D₁ of the fineparticles 10 is preferably larger than the average pore diameter D₂ ofthe porous body 20 so that the fine particles 10 do not enter the deeppart of the pores 29 of the porous body 20. The average particlediameter D₁ of the fine particles 10 is more preferably 1.2 times ormore, further preferably 1.5 times or more, particularly preferably 1.8times or more the average pore diameter D₂ of the porous body 20.

Although the shape of the fine particles 10 is not particularly limited,spherical fine particles are preferred from the viewpoint of equalizingthe accessibility of the protease to the pores 29 of the porous body 20.Further, the fine particles 10 preferably have a uniform averageparticle diameter.

When the average pore diameter of the porous body 20 is about 30 to 150nm, the average particle diameter D₁ of the fine particles 10 ispreferably 100 nm or more, more preferably 150 nm or more. When thesubstrate protein 25 is an antibody and the average pore diameter of theporous body 20 is about 50 nm to 100 nm, the average particle diameterof the fine particles 10 is preferably 120 nm or more, more preferably150 nm or more, particularly preferably 170 nm or more. The upper limitof the average particle diameter D₁ of the fine particles 10 is notparticularly limited, but is preferably 1 μm or less, more preferably500 nm or less, further preferably 300 nm or less, from the viewpoint ofimproving the efficiency of protease proteolysis.

The material of the fine particles 10 is not particularly limited aslong as the protease can be immobilized on the surface thereof, and ametal, a resin, or the like is appropriately used. Alternatively, amaterial obtained by coating the surface of a metal with a resin, amaterial obtained by coating the surface of a resin with a metal, or thelike may be used.

The fine particles 10 preferably have a surface capable of suppressingnonspecific protein adsorption and of selectively immobilizing theprotease thereon. For example, as shown in FIG. 1, fine particles whosesurface is modified by a spacer 11 that can specifically bind with theprotease are appropriately used. The spacer is preferably one that canbind with the protease and does not deactivate the protease.

Further, from the viewpoint of controlling the range of accessibility ofthe protease 15 immobilized on the surface of the fine particles 10, thespacer 11 preferably has a small molecular diameter. The moleculardiameter of the spacer is preferably 5 nm or less, more preferably 3 nmor less, further preferably 2 nm or less. Further, the molecular weightof the spacer is preferably 2000 or less, more preferably 1500 or less,further preferably 1000 or less, particularly preferably 800 or less.The spacer molecule that has a molecular diameter in the above range andis capable of immobilizing the protease is preferably non-protein andpreferably has a functional group such as an amino group, an amidegroup, an ester group, an epoxy group, a carboxyl group, biotin, avidin,or a chelate. For example, the spacer preferably used to immobilizetrypsin has an ester group. Further, a molecule having an activatedester group is also preferably used as the spacer to improve theefficiency of protease immobilization.

In an aspect of the present invention, commercially-available fineparticles modified with a spacer molecule may also be used. For example,fine particles modified with a spacer molecule having an ester groupactivated by N-hydroxysuccinimide are commercially available as fineparticles for affinity purification under the trade name of “FG beadsNHS”.

Preparation of Protease-Immobilized Fine Particles

A method for immobilizing the protease 15 on the surface of the fineparticles 10 is not particularly limited, and an appropriate method canbe adopted depending on the properties of the protease and of the fineparticles (or the spacer molecule modifying the surface of the fineparticles) etc. For example, when trypsin is immobilized on the surfaceof the fine particles modified with the spacer, a suspension of the fineparticles and a solution containing trypsin are mixed together. In thisway, the protease can be immobilized on the surface of the fineparticles.

After the protease is immobilized on the surface of the fine particles,active portions not binding with the protease on the surface of the fineparticles are preferably deactivated. For example, if the spacermolecule not having the protease immobilized thereon is present on thesurface of the fine particles, there is a case where a problem that theunbound spacer molecule binds with an impurity or the like in a sampleso that protease proteolysis is adversely affected, a problem thatpeptide fragments produced by protease proteolysis are immobilized onthe fine particles, or the like occurs. Such a problem is suppressed byblocking the unbound spacer after the immobilization of the protease.The deactivation of the active portions not binding with the protease ispreferably performed by chemical modification. For example, an activatedester group is deactivated by forming an amide bond through a reactionwith an amine.

Protease Proteolysis

The substrate protein is subjected to protease proteolysis by bringingthe porous body 20 having the substrate protein 25 immobilized thereonand the fine particles 10 having the protease 15 immobilized on thesurface thereof into contact with each other in a liquid so that peptidefragments are produced.

In an aspect of the present invention, the condition of the proteaseproteolysis are not particularly limited, and conditions similar tothose of general protease digestion can be appropriately adopted. Forexample, the protease proteolysis is preferably performed by incubationin a buffer solution having a pH adjusted to about the optimum pH valueof the protease at a temperature of usually about 37° C. for about 4hours to 20 hours.

The quantitative mixing ratio between the porous body having thesubstrate protein immobilized thereon and the fine particles having theprotease immobilized on the surface thereof is not particularly limited,either, and may be set so that the amount of the protease becomesappropriate for the amount of the substrate protein. It is to be notedthat protease digestion is generally performed under a condition wherethe ratio (weight ratio) of substrate protein:protease=about 100:1 to20:1. On the other hand, in an aspect of the present invention, theamount of the protease is preferably larger than that used in generalprotease digestion because the access between the substrate protein andthe protease is physically limited due to the combined use of the porousbody and the fine particles. For example, the ratio of substrateprotein:protease is preferably about 30:1 to 3:1, more preferably about15:1 to 4:1, further preferably about 10:1 to 5:1.

In general, when an antibody in a biological sample such as blood issubjected to selective protease digestion, the protease digestion needsto be performed after the sample is first mixed with particles havingProtein G or the like immobilized thereon to immobilize the antibody tothe particles, impurities are removed, and then the antibody is elutedfrom the particles and then denatured with urea or guanidine. In themethod according to the present invention, in contrast, proteaseproteolysis is performed in a state where the antibody is keptimmobilized on the porous body. Further, peptide fragments produced byprotease proteolysis are present in a liquid phase, and thereforepeptide fragments of the Fab domain of the antibody can besite-selectively obtained without performing elution or denaturation ofthe antibody. In this way, in the method according to an aspect of thepresent invention, peptide fragments can be site-selectively recoveredby simpler operation as compared to a conventional method.

Kit for Preparing Peptide Fragments

Peptide fragments may be prepared using a previously-prepared kit forpreparing peptide fragments according to an aspect of the presentinvention. The kit for preparing peptide fragments according to anaspect of the present invention includes a porous body having porescapable of immobilizing a substrate protein and fine particles capableof immobilizing a protease on the surface thereof. The kit may furtherinclude a protease. The fine particles may be provided in a state wherea protease is immobilized on the surface thereof.

FIG. 3 is a diagram showing one embodiment of the kit for preparingpeptide fragments according to an aspect of the present invention. InFIG. 3, fine particles 110 capable of immobilizing a protease on thesurface thereof are provided as a suspension 131. The kit may furtherinclude a protease. The fine particles 110 may be provided in a statewhere a protease is immobilized on the surface thereof. A spin column132 includes an inner container 135 and an outer container 136, and theyare structured so as to be detachably attached to each other. At thebottom of the inner container 135, a porous membrane 120 is providedwhich has pores capable of immobilizing a substrate protein. The porousmembrane 120 has such a pore diameter in order that a liquid isprevented from permeating the porous membrane 120 at an ordinarypressure.

When peptide fragments are prepared using such a spin column, a sample(e.g., a specimen such as blood) containing a substrate protein is firstplaced in the inner container 135 of the spin column to bring the sampleinto contact with the porous membrane. If necessary, the container maybe shaken to bring the sample into uniform contact with the porousmembrane. This operation allows the substrate protein, such as anantibody, to be immobilized in the pores of the porous membrane 120.

The sample liquid after immobilization of the substrate protein on theporous membrane is preferably discharged from the inner container 135.The liquid may be discharged from the opening of the inner container bymanipulation such as pipetting or may be discharged from the bottom ofthe inner container through the porous membrane by centrifugation or thelike. Then, if necessary, washing is performed with an appropriatesolution.

The fine particles 110 having a protease immobilized on the surfacethereof are added to the inner container 135 provided with the porousmembrane 120 having the substrate protein immobilized thereon. Asdescribed above, the protease may previously be immobilized on the fineparticles or may be immobilized on the surface of the fine particlesjust before use.

If necessary, a solution, such as a buffer, may further be added for thepurpose of, for example, optimizing the conditions of proteaseproteolysis. The substrate protein immobilized on the porous membrane120 in the inner container is proteolyzed by the protease immobilized onthe surface of the fine particles 110. As described above, theconditions of protease proteolysis can be appropriately set. Peptidefragments produced by protease proteolysis migrate into the liquidphase.

The peptide fragments produced by site-selectively proteolyzing thesubstrate protein are obtained by recovering the liquid phase afterprotease proteolysis. A method for recovering the liquid phase is notparticularly limited. The liquid phase can be simply recovered bycentrifugation. In this case, the liquid phase is discharged from thebottom of the inner container 135 through the porous membrane andrecovered in the outer container 136. Then, operation such as washing orelution may be performed for the purpose of, for example, elution of thepeptide fragments held in the pores of the porous membrane.

As described above, the use of the kit makes it possible to more simplyperform the operation of preparing peptide fragments according to anaspect of the present invention and to easily automate the operationusing a device. Particularly, trypsin or the like can maintain itsactivity even in a state where it is immobilized on the surface of thefine particles. Therefore, the operation of preparing peptide fragmentscan be further simplified by providing, as the component of the kit, aprotease in a state where it is immobilized on the surface of the fineparticles.

Analysis

A sample containing the peptide fragments obtained above can be analyzedby chromatography or mass spectrometry to identify or quantitate thesubstrate protein. In an aspect of the present invention, the substrateprotein is subjected to site-selective protease treatment, and thereforethe number of types of peptide fragments contained in a sample isreduced. Therefore, the conditions of analysis by mass spectrometry orthe like can be easily set. If necessary, the sample used for analysismay be subjected to pretreatment, such as desalting, solubilization,extraction, concentration, or drying, before analysis.

Mass spectrometry is suitable for identification or quantitation of thesubstrate protein from the peptide fragments produced by proteaseproteolysis. Mass spectrometry can determine the amino acid sequences ofpeptide fragments, and therefore can determine whether or not thepeptide fragments are derived from a specific protein such as anantibody. Further, the concentrations of the peptide fragments in thesample can be determined based on peak intensities.

An ionization method used in mass spectrometry is not particularlylimited, and may be, for example, electron ionization (EI), chemicalionization (CI), field desorption (FD), fast atom bombardment (FAB),matrix-assisted laser desorption ionization (MALDI), or electrosprayionization (ESI). A method for analyzing the ionized sample is notparticularly limited, and may be appropriately determined depending onthe ionization method used. Examples of the method include a magneticdeflection method, a quadrupole (Q) method, an ion trap (IT) method, atime-of-flight (TOF) method, and a Fourier transform ion cyclotronresonance (FT-ICR) method. Alternatively, a triple quadrupole massspectrometer or the like may be used to perform MS/MS analysis or hybridmass spectrometry such as MS³ or higher-order MS.

For the purpose of, for example, more reliably separating the peptidefragments to improve the accuracy of analysis, the sample may beseparated and concentrated by liquid chromatography (LC), solid phaseextraction (SPE), or the like before subjected to mass spectrometry.When the sample is separated by LC, LC/MS including LC prior to massspectrometry may be used so that an eluate from LC is directly ionizedand subjected to mass spectrometry. The sample may be analyzed byLC/MS/MS or LC/MS. that is a combination of LC and tandem massspectrometry. The eluate from LC may be once fractionated beforesubjected to mass spectrometry. A column for LC or a carrier for SPE isnot particularly limited and may be appropriately selected. For example,a hydrophobic column, such as C30, C18, C8, or C4, generally used forpeptide analysis or a carrier for hydrophilic affinity chromatographymay be used.

Existing databases may be used to identify the protein, such as anantibody, based on the result of mass spectrometry. In an aspect of thepresent invention, peptide fragments obtained by site-selective proteaseproteolysis of the substrate protein such as an antibody are used, andtherefore a hit rate in database search or data accuracy is increased.Further, the substrate protein can also be identified by identifying theamino acid sequences of the peptide fragments by hybrid massspectrometry or the like. For example, when the substrate protein is anantibody, the antibody can be identified by determining the sequence ofa peptide fragment containing at least part of the amino acid sequenceof a complementarity-determining region (CDR) having an amino acidsequence specific to the antibody.

When the antibody is detected or quantitated based on the result ofdetection of a specific peptide fragment containing the sequence of CDR,the peptide to be detected preferably has about 5 to 30 amino acidresidues, more preferably about 7 to 25 amino acid residues. If thenumber of amino acid residues is excessively small, the peptide to bedetected is difficult to distinguish from peptide fragments derived fromimpurities or other sites of the same protein, which may cause falsedetection etc. On the other hand, if the number of amino acid residuesis excessively large, in such cases where detection becomes difficult orquantitativity is reduced for the reason that ionization becomesdifficult or the like.

When the concentration of the substrate protein is quantitated, theamount of the substrate protein can be calculated based on the peakareas or peak intensities of detected peptide fragment ions (in the caseof hybrid MS, fragment ions obtained by fragmentation of peptidefragment ions). For example, the concentrations of the peptide fragmentsin the sample are calculated based on the association between apreviously-determined calibration curve and peak areas, the associationbetween peak areas derived from an internal standard added to the sampleand peak areas derived from the sample, or the like, and the amount orconcentration of the substrate protein is calculated based on theconcentration of the peptide fragments.

As described above, according to an aspect of the present invention,both the substrate protein and the protease are immobilized on solidphases to physically control the access between them so that a specificsite in the substrate protein can be subjected to site-selectiveprotease proteolysis. The peptide fragments so obtained can be analyzedby a method such as mass spectrometry, and therefore the protein in thesample can be identified or quantitated without complicated processes.

The method according to an aspect of the present invention isparticularly suitable for detection or quantitation of an antibody. Thesequence or amount of a peptide fragment containing the amino acidsequence of a complementarity determining region can be determined bymass spectrometry of a peptide fragment sample obtained by subjectingthe Fab region of an antibody to selective protease proteolysis.Further, the method according to an aspect of the present invention canbe implemented by simple operation, can ensure reproducibility orquantitativity, and can also be automated. Therefore, the method can beapplied also to fundamental research such as pharmacokinetic analysis,interactive analysis using antigen-antibody reaction, variousinteractome analysis, and identification of immunoprecipitated proteins.In addition, the method according to an aspect of the present inventioncan be expected to be applied to sequencing analysis of biomoleculardrugs such as antibody drugs, quality assurance, confirmation ofidentity of generic drugs, etc.

EXAMPLES

Hereinbelow, experimental examples will be described in which a peptidefragment sample obtained by subjecting human immunoglobulin G (IgG) ortrastuzumab (trade name: Herceptin) to protease proteolysis by themethod according to an aspect of the present invention was subjected tomass spectrometry. It is to be noted that the present invention is notlimited to the following examples.

In the following description, % represents % by weight unless otherwisespecified. Reagents and the like used in the experimental examples areas follows.

Trypsin (sequencing grade, promega)

Lysyl endopeptidase (mass spectrometry grade, Wako Pure ChemicalIndustries, Ltd.)

2-Morpholinoethanesulfonic acid (MES, DOJINDO LABORATORIES)

2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES, DOJINDOLABORATORIES)

Tris(hydroxymethyl)aminomethane (Tris, Wako Pure Chemical Industries,Ltd.)

Reagents and the like other than those listed above, such as organicsolvents, were purchased from Wako Pure Chemical Industries, Ltd.

The following buffer solutions whose pH values were adjusted with aprecise pH meter were used.

MES buffer: 25 mM MES-NaOH, pH 5.5

HEPES buffer: 25 mM HEPES-NaOH, pH 7.0 Ethanolamine buffer: 1Methanolamine-HCl, pH 8.0

Tris buffer: 25 mM Tris-HCl, pH 8.0

<Preparation of Antibody-Immobilized Porous Body>

A suspension of porous resin beads having Protein G bound to thesurfaces thereof (Pierce Biotechnology, Protein G UltraLink resin,average particle diameter: 100 μm, pore diameter: 50 to 100 nm) of 5 μLwas added to 200 μL of MES buffer, and then an antibody solution wasadded thereto. Then, the resulting mixture was gently stirred at roomtemperature for about 1 hour so that an antibody was immobilized bybinding to Protein G on the surfaces of the resin beads. Then, the resinbeads were precipitated by centrifugation at 4° C. (15000 rpm, 1 min) toremove the supernatant. Then, washing with Tris buffer andcentrifugation were repeated twice, and the porous beads were suspendedin Tris buffer. (200 μL). As the antibody solution, a humanimmonglobulin (IgG) solution (10 mg/mL, Sigma-Aldrich) or a trastuzumab(Herceptin, 20 mg/mL, CHUGAI PHARMACEUTICAL CO., LTD.) solution wasused.

<Preparation of Protease-Immobilized Fine Particles>

Nanometer-sized fine particles for protease immobilization (TAMAGAWASEIKI CO., LTD., FG beads NHS) were used which were obtained bymodifying the surfaces of fine particles having an average particlediameter of 190 nm with a spacer whose carboxyl group was activated byN-hydroxysuccinimide (see the following chemical formula, wherein Lrepresents a binding site that binds to the surface of the fineparticles), spacer length: 1 nm).

Isopropanol suspension of FG beads of 50 μL was centrifuged at 4° C.(15000 rpm, 5 min) to precipitate the fine particles and remove thesupernatant. Then, the fine particles were washed with methanol. Asolution containing 50 μg of protease was dissolved in 200 μL of HEPESbuffer, and the resulting solution was added to the fine particles toobtain a suspension in which the fine particles were suspended. Herein,the suspension of the fine particles was performed by ultrasonictreatment for a few seconds to prevent the increase in temperature ofthe suspension.

The fine particle suspension was stirred at 4° C. for 30 minutes andthen centrifuged at 4° C. (15000 rpm, 5 min) to precipitate the fineparticles and remove the supernatant. Then, 200 μL of ethanolaminebuffer was added to suspend the beads, and the resulting suspension wasstirred at 4° C. for 30 minutes to block redundant N-hydroxysuccinimidegroups on the surface of the fine particles with ethanolamine. Then, thefine particles were precipitated by centrifugation at 4° C. (15000 rpm,5 min) to remove the supernatant. Then, washing with Tris buffer andcentrifugation were repeated twice, and the fine particles weresuspended in Tris buffer (100 μL). The protease concentration of thesuspension was 0.5 μg/μL.

Experiment 1 Determination of Amount of Antibody Immobilized on PorousBody

In the preparation of the antibody-immobilized porous body, the amountof the Protein G-binding resin bead suspension per 100 μg of IgG waschanged in the range of 0 to 20 μL, and the resulting supernatant wasanalyzed by SDS-PAGE electrophoresis. The approximate amount of unboundIgG remaining in the supernatant (residual amount of antibody) wasdetermined from the number of pixels per band in the resultingelectrophoretic pattern. The residual amount of antibody tended toreduce as the amount of the Protein G-binding resin beads increased.When the amount of the Protein G-binding resin beads was 10 μL, theresidual amount of antibody was about 3%, from which it was confirmedthat specifications given in the catalog of the Protein G-binding resinbeads were almost reproduced (data not shown).

Experiment 2 Examination of Quantitative Ratio Between Antibody andProtease

The IgG-immobilized porous body suspension (Protein G-IgG) and theprotease-immobilized fine particles (FG beads-Trypsin) were mixedtogether, and the resulting mixture was gently stirred at 37° C. for 15hours to perform protease proteolysis. Then, the resin was precipitatedby centrifugation at 4° C. (15000 rpm, 5 min) to recover the liquidphase (supernatant). The above experiment was performed by changing theamount of the protease-immobilized fine particles so that the amount ofthe protease was 5 μg (Level 1), 10 μg (Level 2), or 25 μg (Level 3). Inthe case of Levels 4 to 6, the experiment was performed in the samemanner except that a porous body on which no IgG was immobilized(Protein G UltraLink resin) was directly used instead of theIgG-immobilized porous body suspension. Further, in the case of Levels 7and 8, only the protease-immobilized fine particles (FG beads-Trypsin)were incubated at 37° C. for 15 hours without using a porous body.

The levels of the above experiments are shown in Table 2. The weight(μg) shown in Table 2 represents the amount of the protein (IgG ortrypsin) in a sample. The SDS-PAGE electrophoretic patterns of theobtained supernatants are shown in FIG. 4. In FIG. 4, the leftmost laneis a molecular weight marker.

TABLE 2 Protein G-IgG FG beads-Trypsin (μg) (μg) 1 100 5 2 100 10 3 10025 4 Protein G only 5 5 Protein G only 10 6 Protein G only 25 7 — 5 8 —10

(Mass Spectrometry)

The supernatants of Levels 1 to 6 were analyzed by MALDI-TOFMS (AXIMAResonance MALDI-QIT TOF MS, SHIMADZU CORPORATION). First,trifluoroacetic acid was added to 20 μL of the supernatant so that itsfinal concentration was 0.5%, and the resulting mixture was purifiedusing a hydrophobic resin-packed tip (Millipore, ZipTip uC18). Then,elution was performed twice with 1 μL of an eluant. The resulting eluatewas directly applied onto a MALDI stainless steel target and air-driedin a clean bench. After the air drying, 1 μL of a 10 mg/mL2,5-dihydroxybenzoic acid solution (DHBA, SHIMADZU GLC Ltd.,water/acetonitrile=50/50) was layered thereon to perform massspectrometry. m/z of the apparatus was calibrated with Angiotensin IIpeptide (m/z=1046.54, Sigma-Aldrich) and ACTH fragment peptide(m/z=2465.20, Sigma-Aldrich). The resulting MS spectra are shown in FIG.5.

All the bands of Levels 4 to 6 shown in FIG. 4 were the same as those ofLevels 7 and 8. Further, all the peaks at m/z=842, 1045, 2211, and 2283detected in the case of Levels 4 to 6 shown in FIG. 5 are derived fromfragments produced by autolysis of trypsin. As can be seen from theseresults, Protein G is not proteolyzed when the protease-immobilized fineparticles and the Protein G-binding porous body are brought into contactwith each other. The reason for this is considered to be that the porediameter of the porous body is smaller than the particle diameter of theprotease-immobilized fine particles, and therefore trypsin immobilizedon the surface of the fine particles cannot have accessibility toProtein G in the pores.

As shown in FIG. 5, in the case of Levels 1 to 3, peaks other than thepeaks of fragments produced by autolysis of trypsin were detected atm/z=835, 913, 1187, 1287, 1510, 1678, and 1946, and were all confirmedto be derived from peptide fragments produced by trypsin proteolysis ofIgG. This result showed that IgG could be selectively proteolyzedwithout proteolyzing Protein G immobilized on the porous body bybringing the IgG-immobilized porous and the trypsin-immobilized fineparticles into contact with each other.

As can be seen from the comparison among the electrophoretic patterns ofLevels 1 to 3 shown in FIG. 4, as the amount of trypsin increases,high-molecular weight bands reduce, that is, the proteolysis reaction ofthe antibody more efficiently proceeds. On the other hand, as the amountof trypsin increases, autolysis becomes more pronounced. Based on theseresults, Level 2 (ratio of substrate:enzyme=10:1) was set as a standardcondition to examine another condition that will be described later.

It is to be noted that protease digestion is generally performed underthe condition where the ratio of substrate protein:protease=100:1 to20:1. In the method according to an aspect of the present invention, theaccessibility between the substrate protein and the protease isphysically limited by the combined use of the porous body and the fineparticles, and therefore the amount of the protease is larger than thatin general protease digestion. For this reason, an optimumsubstrate-enzyme ratio is estimated to be about 10:1 to 5:1.

Experiment 3 Evaluation of Recovered Peptides Based on Proteolysis Time

The IgG-immobilized porous body suspension (amount of IgG on solidphase: 100 μg) and the protease-immobilized fine particles (amount oftrypsin on solid phase: 10 μg) were mixed so that the condition of Level2 selected in Experiment 2 was satisfied, and the time of proteolysis at37° C. was set to (1) 15 mins, (2) 45 mins, (3) 90 mins, (4) 180 mins,(5) 360 mins, and (6) 15 hrs (overnight, O/N). Trypsin proteolysis wasperformed in the same manner as in Experiment 2 except for the abovechange, and obtained samples were subjected to mass spectrometry. Theresulting MS spectra are shown in FIG. 6.

As can be seen from FIG. 6, as the proteolysis time increases, the peaksof peptide fragments increase, that is, the amounts of recovered peptidefragments increase. The recovery rate of peptide fragments was higher inthe case of overnight (6) than in the case of 360 min (5). Particularly,accumulation of fragments easily produced by protease proteolysis atm/z=1187, 1510, 1678, etc. tended to be more pronounced. However, thesefragments are derived from the C region of the antibody, and thereforedo not contribute to the analysis of peptide fragments containing CDR.Therefore, the time of protease proteolysis was set to 6 hrs to make thefollowing study.

Experiment 4 Trypsin Proteolysis and Mass Spectrometry of Trastuzumab

An antibody-immobilized porous body was prepared using Herceptin insteadof IgG as an antibody and subjected to trypsin proteolysis under thecondition selected in Experiments 2 and 3, that is, under the conditionwhere the amount of the protease-immobilized fine particles per 100 μgof the amount of antibody on solid phase was 10 μg and the proteolysistime was 6 hours. Then, mass spectrometry was performed and database(Mascot server) analysis was performed based on the result of massspectrometry. As can be seen from FIG. 7, Herceptin (Chain B, X-RayStructures Of The Antigen-Binding Domains From Three Variants OfHumanized Anti-P185-Her2 Antibody 4d5 And Comparison With MolecularModeling) is identified with a very high score.

In order to confirm that peptide fragments of Herceptin were detected bymass spectrometry, a more detailed analysis was performed in thisexperiment. FIG. 8 shows the resulting mass spectrum (MALDI-TOFMS).

Further, the resulting supernatant after proteolysis was analyzed byLC-MS (LCMS-8080 Triple-quadrupole ultra high-performance liquidchromatography-MS, SHIMADZU CORPORATION). FIG. 9 shows the resultingLC-MS chromatogram and FIG. 10 shows the resulting MS spectra. LC-MSmeasurement was performed using a sample prepared by adding formic acidto the supernatant to a final concentration of 0.5%, and LC wasperformed under the following conditions.

<HPLC Solutions>

Solution A: 0.1% formic acid, 1% acetonitrile/aqueous solution

Solution B: 0.1% formic acid, acetonitrile solution

<Column>

ShimPack ODS XR-ODS II (inner diameter: 2 mm, column length: 50 mm)

Column Temperature: 40° C.

Flow Rate: 0.4 mL/min

Injection Amount: 20 μL

<Gradient Program>

0-2 min: % B=0

2-10 min: % B=0-40 gradient

10-11 min: % B=40-98 gradient

11-13 min: % B=98

13-13.5 min: % B=98-0 gradient

13.5-15 min: % B=0

Underlined peptide sequences in the heavy chain (FIG. 11A, SEQ ID No. 1in Sequence Listing) and light chain (FIG. 11B, SEQ ID No. 2 in SequenceListing) of trastuzumab were detected and identified by the above massspectrometry. As can be seen from FIG. 11A, all the CDR1 (SEQ ID No. 3in Sequence Listing), CDR2 (SEQ ID No. 4 in Sequence Listing), CDR3 (SEQID No. 5 in Sequence Listing) of the heavy chain were detected. Further,as shown in FIG. 11B, the CDR1 (SEQ ID No. 6 in Sequence Listing) andCDR2 (SEQ ID No. 7 in Sequence Listing) of the light chain weredetected. A tryptic fragment containing the sequence of CDR3 (SEQ ID No.8 in Sequence Listing) of the light chain has a length of 4 amino acidresidues or 37 amino acid residues, and is therefore not suitable formass spectrometry. This is the reason that the CDR3 of the light chaincould not be identified. However, it is considered that a peptidefragment that allows the CDR3 of the light chain to be detected by massspectrometry can be prepared by changing the type of protease used.Further, in this experiment, 5 out of the total 6 CDRs of the heavy andlight chains were detected, although the CDR3 of the light chain couldnot be detected. Therefore, as shown in FIG. 7, trastuzumab wasidentified by database analysis.

As can be seen from the above, since the peptide fragments prepared bythe method according to an aspect of the present invention are peptidefragments obtained by site-selective protease proteolysis of theantibody, the antibody can be identified by mass spectrometrymeasurement without the need for setting complicated measurementconditions.

Experiment 5 Study of Mixed Protease Proteolysis

In order to study the applicability of mixed protease proteolysis to asystem according to an aspect of the present invention, the experimentof protease proteolysis was performed using trypsin and lysylendopeptidase (Lys-C) in combination.

An antibody (IgG or Herceptin) was subjected to protease proteolysis inthe same manner as in each of the above experimental examples bystirring an antibody-immobilized porous body having 100 μg of theantibody immobilized on Protein G bound thereto and fine particleshaving 10 μg of protease immobilized thereon at 37° C. for 6 hours. Theexperiment was performed for both cases where IgG was used as anantibody and where Herceptin was used as an antibody under the conditionwhere the ratio (weight ratio) between trypsin and lysyl endopeptidasewas set to (1) 10:0, (2) 9:1, (3) 8:2, and (4) 0:10. The resultingsupernatant after proteolysis and the component immobilized on thesurface of the porous body were analyzed by SDS-PAGE electrophoresis.The resulting electrophoretic patterns are shown in FIG. 12. In FIG. 12,the leftmost lane is a molecular weight marker.

The supernatants after proteolysis of Levels 1 to 4 obtained whenHerceptin was used as an antibody were subjected to mass spectrometry inthe same manner as in the above Experiment 2. The resulting MS spectraare shown in FIG. 13. Further, database analysis was performed by Mascotserver in the same manner as in Experiment 4 based on the result of massspectrometry. The database analysis result of Level 1 (trypsin 100%) wasthe same as that obtained in the above Experiment 4 (FIGS. 7 and 8). Thedatabase analysis results of Levels 2 to 4 are as follows (data was notshown).

-   -   <Level 2: trypsin lysyl endopeptidase=90:10>        Mixture        gi|442924 Mass: 23708 Score: 117 Expect: 4.9e−007 Matches: 13        Chain B, X-Ray Structures Of The Antigen-Binding Domains From        Three        Variants Of Humanized Anti-P185-Her2 Antibody 4d5 And Comparison        With Molecular Modeling        gi|184747 Mass: 36012 Score: 64 Expect: 0.11 Matches: 10        immunoglobulin gamma-1 heavy chain constant region (Homo        sapiens)    -   <Level 3: trypsin lysyl endopeptidase=80:20>        Mixture        gi|442924 Mass: 23708 Score: 115 Expect: 7.8e−007 Matches: 13        Chain B, X-Ray Structures Of The Antigen-Binding Domains From        Three        Variants Of Humanized Anti-P185-Her2 Antibody 4d5 And Comparison        With        Molecular Modeling        gi|184747 Mass: 36012 Score: 54 Expect: 1.1 Matches: 9        immunoglobulin gamma-1 heavy chain constant region (Homo        sapiens)    -   <Level 4: lysyl endopeptidase 100%>        gi|184747 Mass: 36012 Score: 71 Expect: 0.021 Matches: 8        immunoglobulin gamma-1 heavy chain constant region (Homo        sapiens)

In the cases of Levels 2 and 3 in which trypsin and lysyl endopeptidasewere used in combination, the analysis result showed that the constantregion of a human-derived antibody as well as the antigen-binding regionof Herceptin were detected. In the case of Level 4 in which lysylendopeptidase was used alone, the analysis result showed that Herceptinwas not detected, and only the constant region of an antibody wasdominantly detected.

As can be seen from the electrophoretic patterns shown in FIG. 12, thereis a tendency that as the ratio of lysyl endopeptidase increases, thenumber of peptide fragments in the supernatant and the areas of bandsalso increase, that is, proteolysis efficiency and peptide recovery rateincrease. Further, as shown in FIG. 13, the types or amounts of peptidefragments detected increase as the ratio of lysyl endopeptidaseincreases. However, most of the peptide fragments newly detected in thecase of Levels 2 to 4 are derived from the Fc domain of Herceptin, fromwhich it is found that the site-selectivity of protease proteolysis(i.e., the property of selectively proteolyzing the Fab domain) isreduced.

As can be seen from these results, as the amount of lysyl endopeptidaseused increases, the proteolysis efficiency of the antibody increases,but the site-selectivity of protease proteolysis reduces, and thereforethe relative production amount of peptide fragments from the V regionreduces as the production amount of peptide fragments from the constantregion increases so that the accuracy of detection and identification ofthe antibody tends to reduce. Therefore, from the viewpoint ofsite-selectively proteolyzing the Fab region of the antibody andspecifically detecting CDR by the method according to an aspect of thepresent invention, trypsin is preferably used alone. When trypsin andlysyl endopeptidase are used in combination, the amount of lysylendopeptidase mixed is preferably 10% or less.

It is to be noted that site-selective proteolysis was not performed inthe case of Level 4 using lysyl endopeptidase alone, whereas CDRs wereefficiently detected by database analysis in the case of Level 1(Experiment 4) using trypsin alone, from which it was found that the Vregion of the Fab domain was subjected to selective proteaseproteolysis. It can be said that the above results showed that when thesubstrate protein was an antibody, the steric access of the protease tothe antibody was appropriately controlled by applying an aspect of thepresent invention using trypsin so that site-selective proteaseproteolysis could be achieved.

Further, this experiment showed that when trypsin and lysylendopeptidase were used in combination, each of the proteases did notlose its function and could proteolyze the substrate protein immobilizedin the pores. An antibody has a V region at the end of its molecule, andtherefore when the amount of used lysyl endopeptidase increases,site-selectivity tends to reduce. However, it was suggested that in thecase where another protein was used as a substrate, there was apossibility that site-selectivity or proteolysis efficiency was improvedby using a combination of proteases.

As can be seen from the above experimental examples, according to anaspect of the present invention, a peptide fragment sample can beobtained by subjecting a protein, such as an antibody, to site-selectiveprotease proteolysis by a simple method, and the obtained peptidefragment sample is suitable for identification or detection of theprotein by mass spectrometry.

The inventors have intensively studied to find that immobilization ofboth a substrate protein, such as an antibody, and a protease on solidphases makes it possible to achieve site-selective protease proteolysisof the substrate protein.

One aspect of the present invention relates to a method for preparingpeptide fragments by proteolyzing a protein with a protease. The methodincludes a step of proteolyzing a substrate protein with a protease bybringing a porous body in which a substrate protein to be proteolyzed isimmobilized in pores thereof and fine particles having a proteaseimmobilized on surface thereof into contact with each other in a liquid(proteolysis step). The porous body on which the substrate protein to beproteolyzed is immobilized in the pores thereof can be obtained by astep of immobilizing a substrate protein to be proteolyzed in pores of aporous body (substrate immobilization step). In an aspect of the presentinvention, an average particle diameter of the fine particles ispreferably larger than an average pore diameter of the porous body.

When the particle diameter of the fine particles is larger than theaverage pore diameter of the porous body, the protease immobilized onthe surface of the fine particles can access the opening portions of thepores of the porous body and their vicinities (the interface between theporous body and the liquid phase and its vicinity), but cannot accessthe deep parts of the pores. In this way, the accessible region of theprotease is physically (spatially) limited, and therefore the proteaseselectively accesses a specific site of the substrate proteinimmobilized in the pores of the porous body. This makes it possible toachieve site-selective protease proteolysis (fragmentation) of thesubstrate protein.

In an aspect of the present invention, a predetermined region of thesubstrate protein is preferably immobilized on the porous body. In thisembodiment, the region immobilized on the porous body is located in thedeep parts of the pores so that a region different from the immobilizedregion is located near the opening portions of the pores. When a regiondifferent from the selective proteolysis site of the substrate protein,i.e., a region preferred not to be subjected to protease proteolysis, isimmobilized on the porous body, the protease immobilized on the surfaceof the fine particles accesses the selective proteolysis site of thesubstrate protein located near the opening portions of the pores so thatprotease proteolysis is performed. This allows site-selective proteaseproteolysis at a desired site in the substrate protein.

A linker molecule capable of site-specific interaction with thesubstrate protein is preferably immobilized in the pores of the porousbody. The substrate protein is immobilized in the pores of the porousbody preferably through the linker molecule. Examples of the linkermolecule used when the substrate protein is an antibody include ProteinG and Protein A. Such a linker molecule site-specifically binds with theFc region of the antibody, and therefore the Fc region of the antibodyis immobilized on the porous body so that the Fab region of the antibodyis located near the opening portions of the pores. According to thisembodiment, the Fab region of the antibody can be subjected to selectiveprotease proteolysis.

When the linker molecule is immobilized in the pores of the porous body,a molecule in which the linker molecule binds with the substrate proteinpreferably has a size 0.5 times to 1.5 times the average pore diameterof the porous body. Such molecular size adjustment makes it possible toincrease the probability of access of the protease immobilized on thesurface of the fine particles to the selective cleavage site of thesubstrate protein, thereby improving the site-selectivity of proteaseproteolysis.

It is preferred that the surface of the fine particles is modified witha spacer molecule capable of binding with the protease, and the proteaseis immobilized on the surface of the fine particles through the spacermolecule. The immobilization of the protease through the spacer moleculemakes it possible to suppress the detachment of the protease from thesurface of the fine particles, thereby improving the site-selectivity ofprotease proteolysis. Further, the adjustment of molecular size of thespacer makes it possible also to allow the protease to selectivelyaccess a desired position in the substrate protein to improve thesite-selectivity.

In an aspect of the present invention, the protease to be immobilized onthe surface of the fine particles is preferably trypsin or a combinationof trypsin and another protease. When the substrate protein is anantibody, trypsin is preferably used alone, or in a combination ofproteases. When proteases are used in a combination, the amount oftrypsin is preferably 90% or more of the total amount of proteases.Particularly, when the substrate protein is an antibody, the Fab domaintends to be subjected to selective protease proteolysis by using trypsinso that the protease proteolysis of the Fc domain is suppressed.

The average pore diameter of the porous body is preferably about 30 to150 nm, and the average particle diameter of the fine particles ispreferably 100 nm or more. Particularly, when the substrate protein isan antibody and the average pore diameter and the average particlediameter are within the above ranges, the Fab region can be morereliably site-selectively proteolyzed.

Another aspect of the present invention relates to a kit for peptidefragment preparation used for the above method. The peptide fragmentpreparation kit according to an aspect of the present invention includesa porous body having pores capable of immobilizing a substrate protein,and fine particles capable of immobilizing a protease on surfacethereof. The fine particles may be provided in a state where theprotease is immobilized on the surface thereof. The porous body of thekit and a sample (e.g., a specimen such as blood) are brought intocontact with each other such that the subject substance (substrateprotein such as an antibody) in the sample can be immobilized in thepores of the porous body. The substrate protein is subjected tosite-selective protease proteolysis by bringing the porous body afterimmobilization of the substrate protein and the fine particles havingthe protease immobilized on the surface thereof into contact with eachother in a liquid.

Peptide fragments obtained by the above method are analyzed by massspectrometry or the like, which makes it possible to detect (identify)or quantitate the substrate protein. In the present invention, thesubstrate protein is subjected to site-selective proteolysis, andtherefore the number of types of peptide fragments contained in ameasurement sample can be significantly reduced. Therefore, the settingof measurement conditions of mass spectrometry can be simplified, andthe accuracy of analysis is also expected to be improved.

For example, when the substrate protein is an antibody, the methodaccording to an aspect of the present invention can achievesite-selective protease proteolysis of the Fab region containing acomplementarity determining region, and therefore a peptide fragmentcontaining at least part of the sequence of complementarity determiningregion of the antibody can be produced as a detection target. Thecomplementarity determining region has an amino acid sequence specificto each antibody. Therefore, the antibody can be detected or quantitatedby analyzing the peptide fragment containing the sequence of thecomplementarity determining region.

According to an aspect of the present invention, a protein, such as anantibody, can be subjected to site-selective protease proteolysis by asimple method to obtain peptide fragments. When the method according toan aspect of the present invention is applied to an antibody,proteolysis of Fc region of the antibody is suppressed, and the Fabregion containing CDR is subjected to selective protease proteolysis,and therefore the concentration of the peptide fragment containing theamino acid sequence of CDR, which is important for identification of theantibody, in a sample is increased.

The method according to an aspect of the present invention makes itpossible to significantly reduce the number of types of peptidescontained in a measurement sample. Therefore, the setting of conditionsof mass spectrometry can be simplified, and the accuracy of analysis canalso be expected to be improved. The concentration of an antibody drugin blood can also be quantitated by analyzing obtained peptidefragments. Therefore, the method according to an aspect of the presentinvention can also be applied as a pretreatment method for a system formeasuring the concentration of an antibody drug in a preclinical orclinical trial.

Further, the method according to an aspect of the present invention canbe applied not only to antibody drugs but also to many proteins, andtherefore can be expected to be extensively applied to pharmaceuticalindustry. In addition, the method according to an aspect of the presentinvention can also be expected to be applied, for example, in the fieldof fundamental research such as interactive analysis of biomolecules.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

What is claimed is:
 1. A kit for proteolyzing a protein, comprising: atleast one porous body having a plurality of pores in which a protein isto be immobilized; and a plurality of particles having an averageparticle diameter larger than an average pore diameter of the pores,wherein the particles have a surface modified by a spacer such that aprotease has been immobilized or is to be immobilized on the surface byspecific binding with the spacer, a ligand molecule is immobilized inthe pores of the porous body, the ligand molecule is capable ofsite-specifically interacting with and controlling an orientation of theprotein to be immobilized, and the average particle diameter or theaverage pore diameter is sized such that each of the pores has a limitto the region accessible to the protease, and that the protein is to beimmobilized with a tip portion extending into the region accessible tothe protease.
 2. The kit of claim 1, wherein the particles have aprotease immobilized on the surface of the particles modified by thespacer.
 3. The kit of claim 1, wherein the average particle diameter ofthe particles is 100 nm or greater and the average pore diameter of thepores in the porous body is in a range of from 30 nm to 150 nm, providedthat the average particle diameter is larger than the average porediameter.
 4. The kit of claim 1, wherein the ligand molecule is capableof site-specifically interacting with Fc domain of an antibody.
 5. Thekit of claim 1, wherein the ligand molecule is at least one selectedfrom the group consisting of protein A and protein G.
 6. The kit ofclaim 1, wherein the spacer allows specific binding with the proteasewithout deactivating the protease.
 7. The kit of claim 1, wherein thespacer has a molecular diameter of 5 nm or less.
 8. The kit of claim 1,wherein the spacer has a molecular diameter of 2 nm or less.
 9. The kitof claim 1, wherein the spacer has a molecular weight of 2000 or less.10. The kit of claim 2, wherein the spacer has a molecular weight of 800or less.
 11. The kit of claim 1, wherein the spacer has one of an aminogroup, an amide group, an ester group, an epoxy group, a carboxyl group,biotin, avidin, and a chelate.
 12. The kit of claim 1, wherein thespacer has an ester group.
 13. The kit of claim 1, wherein the spacerhas an activated ester group.
 14. A kit for proteolyzing a protein,comprising: at least one porous body having a plurality of pores inwhich a protein is immobilized; and a plurality of particles having anaverage particle diameter larger than an average pore diameter of thepores, wherein the particles have a surface on which a protease has beenimmobilized, a ligand molecule is immobilized in the pores of the porousbody, the ligand molecule immobilized in the pores is capable ofsite-specifically interacting with the immobilized protein, thereby anorientation of the protein immobilized in the pores is controlled, theaverage particle diameter or the average pore diameter is sized suchthat the protease immobilized on the surface of the particles has accessto the protein immobilized in the pores of the porous body, the poreseach have a limit to the region accessible to the protease, and theprotein immobilized in the pores has a tip portion extending into theregion accessible to the protease.